Endowing homodimeric carbamoyltransferase GdmN with iterative functions through structural characterization and mechanistic studies

Iterative enzymes, which catalyze sequential reactions, have the potential to improve the atom economy and diversity of industrial enzymatic processes. Redesigning one-step enzymes to be iterative biocatalysts could further enhance these processes. Carbamoyltransferases (CTases) catalyze carbamoylation, an important modification for the bioactivity of many secondary metabolites with pharmaceutical applications. To generate an iterative CTase, we determine the X-ray structure of GdmN, a one-step CTase involved in ansamycin biosynthesis. GdmN forms a face-to-face homodimer through unusual C-terminal domains, a previously unknown functional form for CTases. Structural determination of GdmN complexed with multiple intermediates elucidates the carbamoylation process and identifies key binding residues within a spacious substrate-binding pocket. Further structural and computational analyses enable multi-site enzyme engineering, resulting in an iterative CTase with the capacity for successive 7-O and 3-O carbamoylations. Our findings reveal a subclade of the CTase family and exemplify the potential of protein engineering for generating iterative enzymes.

-It might be illustrative to show a superposition (with the rmsd calculation) of the 2 subunits of GdmN in the asymmetric unit.
-page 12, line 212: It is said that the enzyme contains and iron ion. Perhaps the authors could briefly explain the evidence of the presence of this ion. Distances and geometry of coordination sphere? Similarity with TobZ or other enzymes? Perhaps figure S14 could be completed with the electron density map in the iron coordination sphere.
- Figure 3. Perhaps indicating the position of the iron ions with a sphere of different color will help to better identify the location of the two active sites in the dimer.
-page 15, lane 260-261. "The movements of the side chains of V24 and F25 resulted in better shape complementarity to accommodate 1". In figure 4, these movements appear very small, perhaps less than 1 Å. Perhaps the authors might want to specify the distance between the position of the C-alphas of the residues in the two structures to give a better sense of this movement.
-I am curious about the role of Mg2+ in the enzyme reaction. The concentration of MgCl2 for crystallization is 1.5 mM, which seems small for the combination with 1.5 mM ATP and 1.5 mM CP. In contrast, for the activity assays, the MgCl2 concentration is 10 mM. Why is Mg2+ important? Why the structures do not show the bound ion?
- Figure S19, legend. It is not clear what the authors mean with "Stereo" 2Fo-Fc maps....
-The authors might want to consider changing the order of the figures in the supplementary material to follow the order in which they appear in the text. For instance, figure S10 appears before than figures S6-9; S13 is mentioned earlier than S11 and S12; S21 appears before than S20; and S35 and S36 appear before than S29-34. I also suggest to merge figures S11 and S13, perhaps eliminating panel S13a, since it already shows in Fig. 3. Also, table S4 appears in the text before than tables S1-3.
Reviewer #2 (Remarks to the Author): In this work, a carbamolytransferase GdmN was engineered based on several holo-crystal structures to gain iterative function of successive 7-O and 3-O carbamoylation, in which hydrogen bonding interation and loop flexibility were found to be critical to the regioselectivity. The manuscript is overall well organized and supported by solid data. There are several points to be clarified.
1. Based on the crystal structures obtained, MD simulation would be an effective approach for elucidating the catalytic activity and mechanisms. Did authors performed any MD? for example, compound 3 and GdmN tetra mutant (Fig S33).
2. How about the effect of key residues identified in this study on other CTases? it also would be interesting to interrogate the corresponding residues in Asm21.
This manuscript describes some interesting enzyme "redesign" but there are major issues that must be addressed before publication. 1) Why are the authors convinced that they have an X-ray structure of a tetrahedral intermediate? The 2Fo-Fc omit maps in Figure S19 are not at all convincing.
2) How do the authors know that tetrahedral intermediate decomposition is rate-limiting? I see no convincing evidence of that.
3) The process for making this catalyst *iterative* does not seem rational to me. Why should readers believe it was not an accidental discovery? What are the principles that make a catalyst "iterative" and how were those used in the *design* of mutants? 4) No references are provided for DFT computational methods. Also, the barriers reported are all too high for biological conditions for the ring closing nucleophilic attack examined.

Response to Reviewers' Comments
We sincerely thank the reviewers for the insightful comments and constructive suggestions to improve the quality of our manuscript. We do agree with all comments from reviewers and have carefully addressed all the reviewers' comments. We provide a point-by-point response to all comments from reviewers in BLUE font below (the line numbers and figure numbers refer to the revised manuscript unless otherwise stated). Moreover, the revised contents are in RED, and AMP-CP, the previous abbreviation for carbamoyladenylate, was accurately changed to carbamoyl-AMP throughout the manuscript.

Reviewer #1 (Remarks to the Author):
This is a thorough and very well written study addressing the rational redesign of GdmN to become an iterative enzyme capable of catalyzing two consecutive carbamoylations on a single substrate, as a strategy to expand the potential and increase the efficiency of synthetic applications. In particular, they aimed for an enzyme capable of performing successive 7-O and 3-O carbamoylations of maytansinoids derivatives. Previously, they had described that Asm21, a carbamoyl transferase within the GdmN group, was capable of a dual carbamoylation during ansamitocin synthesis. The authors undertake an extensive and in-depth structural and functional characterization of these enzymes that will allow them to successfully redesign the active site and broaden the catalytic capabilities. the increased plasticity of the reprogrammed active site. Finally, sequence analysis and homology modeling reveal residues in this subgroup of carbamoyltransferases that could account for they different specificity and that could be altered to increase substrate flexibility. The authors also perform computational analysis for the cyclization of the product and for its binding to the mutated enzyme, which shows that they do not leave any loose ends in the story, but I cannot comment on this computational analysis because is not my expertise.
Overall, the article compiles an impressive amount of work that combines many different and sound methodologies. The results are solid, clearly described and exhaustively documented (very extensive supplementary material). The conclusions of the manuscript are well supported by the data and analysis of the authors. I strongly support the acceptance and publication of the article, and only have some minor questions/suggestions: -page 6, line 107: In this sentence, for clarity, the authors could specify that they refer to Class III CTases: "The holoenzymes of all characterized (class III) CTases..."

Response:
We have changed the sentence to "The holoenzymes of all characterized (class III) CTases..." as suggested.

Response:
We have changed all "FPLC" to "size-exclusion chromatography" as suggested.
-It might be illustrative to show a superposition (with the rmsd calculation) of the 2 subunits of GdmN in the asymmetric unit.  -page 15, lane 260-261. "The movements of the side chains of V24 and F25 resulted in better shape complementarity to accommodate 1". In figure 4, these movements appear very small, perhaps less than 1 Å. Perhaps the authors might want to specify the distance between the position of the C-alphas of the residues in the two structures to give a better sense of this movement.

Response:
We have specified the distance between the position of the side chains of the residues in the two structures in Figure 4 in the revised manuscript.

Response:
We have replaced one-letter codes with three-letter codes of amino acids in the revised manuscript as suggested.
-I am curious about the role of Mg 2+ in the enzyme reaction. The concentration of MgCl2 for crystallization is 1.5 mM, which seems small for the combination with 1.  Figure S17). The structural superposition revealed that the site coordinating the Mg 2+ ion is highly conserved, with conservation of residues corresponding to S533 in GdmN and S530 in TobZ. Further substituting S533 with Ala abolished the catalytic activity. The above experimental results determined that Mg 2+ ion is important.
In the previous study 2 , we have examined different concentrations of different metal ions on the catalytic activity, and found that the best condition is 10 mM MgCl2.
However, when we carried out crystallization experiments, we found that 10 mM

Response:
We are sorry for this. We referred to Figures S4-S6 in the literature 1 , and intended to mean the stereo view of 2Fo-Fc maps. Thanks to reviewer's reminder, we referred to other literatures 6,7 , and found that this word is not suitable. In order to avoid confusion, we have removed "Stereo" in the revised supplementary material.
-The authors might want to consider changing the order of the figures in the supplementary material to follow the order in which they appear in the text. For instance, figure S10 appears before than figures S6-9; S13 is mentioned earlier than S11 and S12; S21 appears before than S20; and S35 and S36 appear before than S29-34. I also suggest to merge figures S11 and S13, perhaps eliminating panel S13a, since it already shows in Fig. 3.
Also, table S4 appears in the text before than tables S1-3.

Response:
We have changed the order of the figures and tables in the revised supplementary material as suggested.

Reviewer #2 (Remarks to the Author):
In this work, a carbamolytransferase GdmN was engineered based on several holocrystal structures to gain iterative function of successive 7-O and 3-O carbamoylation, in which hydrogen bonding interaction and loop flexibility were found to be critical to the regioselectivity. The manuscript is overall well organized and supported by solid data. There are several points to be clarified. 2. How about the effect of key residues identified in this study on other CTases? it also would be interesting to interrogate the corresponding residues in Asm21.

Response: Thanks for the suggestions.
We have carried out sequence alignment and homology modeling of other class III Figure S4 and S39). We found that the active site histidine (His27 in GdmN corresponds to His27 in Asm21) is highly conserved. The residues responsible for the formation of carbamoyl-adenylate (carbamoyl-AMP) are highly conserved, with conservation of residues corresponding to K443/M476/S533 in GdmN and K446/M479/S536 in Asm21. The "keystone" tyrosine, which stabilizes the substrate binding pocket (Y82 in GdmN corresponds to Y82 in Asm21) is highly conserved across class III CTases. On the basis of biochemical experiments and crystallographic studies in our study, we preliminarily speculated that these residues play similar roles in other CTases. Figure S1, these CTases recognize different substrates. Further sequence alignment of substrate binding pocket revealed subtle alterations in "plasticity residues" (Supplementary Figure S39), which may contribute to the considerable substrate flexibility of these CTases. It will be intriguing to decipher whether multi-site mutagenesis of these residues in Asm21, Asc21b, and other CTases, could repurpose substrate flexibility. We will undertake further structure-guided engineering and crystallographic studies of Asm21, Asc21b, and other CTases, thereby better interpreting the effect of these residues on other CTases.

As shown in Supplementary
3. What is the reaction time of Fig 6? will 3 be completely converted to 4 in the end?
Response: The activity assays of GdmN tetra mutant were performed at 30 ℃ for 24 h, to yield 3:4 in roughly a 2:1 ratio at 39.85% conversion, as mentioned in the legend of Figure 6. We have extended the reaction time of this reaction system, while compound 3 could not be completely converted to compound 4. When we reduced the concentration of compound 1 to 0.1 mM and kept other conditions unchanged, compound 1 could be completely converted to compound 4 (Figure r2). In order to keep the reaction system of the study consistent and show the successive reaction process, we have put the results in Figure 6.

Line 458 Y82 and F82?
Response: Sorry for this, we have corrected it in the revised manuscript.

Reviewer #3 (Remarks to the Author):
This manuscript describes some interesting enzyme "redesign" but there are major issues that must be addressed before publication. 1) Why are the authors convinced that they have an X-ray structure of a tetrahedral intermediate? The 2Fo-Fc omit maps in Figure S19 are not at all convincing.
Response: Thanks for the suggestions. The Fo-Fc omit maps of different ligands have been added in Supplementary Figures S15, S18, and S34. We also re-drawn the 2Fo-Fc maps of different ligands for clarity in Supplementary Figures S16, S19,   3) The process for making this catalyst *iterative* does not seem rational to me.
Why should readers believe it was not an accidental discovery? What are the principles that make a catalyst "iterative" and how were those used in the *design* of mutants?
Response: We understand the concern from the reviewer. In recent years, some iterative enzymes were reported, such as methyltransferases 9 , glycosyltransferases 10-12 , and etc. However, the transition processes from canonical enzymes to iterative enzymes remain poorly understood. To explore the processes, some researchers attempted to utilize protein engineering to repurpose canonical enzymes into iterative enzymes in recent years 13 . Xiaojing Wang and coworkers evaluated two orthologous Omethyltransferases (58% identity), which modify the same substrate to afford orthogonal regioisomeric outcomes. Based on protein homology modeling, they proposed that subtle alternations of residues lining the substrate binding pocket force the substrates to adopt different binding poses. Subsequently, structure-guided engineering of these "plasticity residues" showed that site-specific replacement of these residues successfully reprogramed the regioselectivity of the enzyme to create iterative mutants.
Inspired by these studies, we examined ansamycins and their corresponding CTases (Supplementary Figure S1). During ansamitocin biosynthesis, Asm21 could catalyze dual carbamoylation, utilizing both a polyketide backbone and a glycosyl moiety as substrates, suggesting that this subclade of CTases might contain a spacious binding pocket and hold promising potential for redesign. In the biosynthesis of ansacarbamitocins, there are two carbamoyltransferase genes, asc21a and asc21b, responsible for the three carbamoylation modifications, and Asc21b was proved to catalyze 3-O-carbamoylation of 3 14 . In our study, we found that GdmN could catalyze 1 to generate 3. Similar to two orthologous O-methyltransferases in the abovementioned studies, GdmN and Asc21b share high sequence identity (61%).
Therefore, we performed structural analysis and structure-guided mutagenesis to reprogram the regioselectivity of GdmN. Coupled with homology modeling of Asc21b, we examined differences of substrate-binding pocket of GdmN and Asc21b, and further performed structure-guided mutagenesis, thereby expanding the substrate scope of GdmN.
Although these successful studies might provide some insights into the transition processes between canonical enzymes and iterative ones, the principles for design of iterative catalysts still required more examples. We will undertake further researches to explore whether structural analysis of enzymes with orthologous regioselectivity and subsequent structure-guided active site engineering can be successfully applied in protein engineering of other iterative enzymes.  Figures S22 and S23). Our calculations showed that this conversion of compound 2 in water is relatively facile, with a Gibbs free energy barrier of 15.3 kcal/mol. Based on these results, we have revised the manuscript, and detailed calculation results have been added to the revised supplementary material. Besides, we have cited references 33,34, and 71-76 in the revised manuscript.